Peptides and other molecules which inhibit the assembly of the hepatitis B virus, methods of treatment, and pharmaceutical compositions comprising them.
The present invention relates to peptide compositions specific for the diagnosis, treatment or prevention of hepatitis B virus infection.
Hepatitis B virus (xe2x80x9cHBVxe2x80x9d) infects human at a very high rate. It is estimated that at least about 300 million people are chronic carriers of HBV. Despite extensive research, additional safe and effective therapies remain to be identified.
HBV infections thus continue to represent a major public health problem worldwide. Infection with the virus results in a gamut of clinical symptoms ranging from minor flu-like symptoms to death. Available vaccines produced from the serum of HBV carriers do not provide the appropriate means to control and eradicate the disease worldwide because of limited resources and production costs involved. Vaccines produced based upon recombinant DNA technology overcome some of these disadvantages, however, there is still a need for additional means to control and eradicate the HBV virus.
The biology, structure and immunochemistry of HBV and the genetic organization of its DNA genome are known. Ganem, D., Varmus, H. E., Ann. Rev. Biochem. 56: 651-693 (1987). The virus is transmitted by three general mechanisms: (1) by inoculation with infected blood or body fluids, (2) by close family or sexual contact, or (3) by infection during pregnancy, where the mother transmits the virus to her child. HBV consists of a nucleocapsid, a small 3.2-kb DNA genome, and the viral polymerase enclosed by the core antigen of the virus, surrounded in turn by the HBV surface antigen (HBsAg). The viral envelope contains three different, but related HBsAg polypeptides, which overlap extensively from their carboxyl termini and arise from variable use of initiation triplets at different points within a continuous open reading frame. The long polypeptide (L polypeptide) is the product of the entire reading frame and comprises the pre-S 1 domain of 108 amino acids (or 119, depending on virus subtype) at its amino terminus, followed by the pre-S2 domain of 55 amino acids, and the short polypeptide (S polypeptide) region of 226 amino acids. The medium-length polypeptide (M polypeptide) has the pre-S2 domain at its amino terminus followed by the S region, whereas the S polypeptide, which is the most abundant form, consists of only the S region. The pre-S regions are believed to play a role in both viral assembly and attachment to the host cell. The S form is more abundant than the M and L forms of HBsAg in the virus, and occurs in both glycosylated and nonglycosylated forms. In addition to its presence in the viral envelope, HBsAg is found in large quantities in the serum of infected individuals as both spherical and filamentous particles, and proportions of the L, M and S polypeptides in these three forms varies appreciably.
The immunologic markers of HBV infection include the surface antigen (HBsAg), the core antigen (HBcAg), the xe2x80x9cexe2x80x9d antigen (HBeAg) and their respective antibodies as well as virus polymerase and x antigen (xe2x80x9cHBxAgxe2x80x9d). Antibodies against HBsAg are protective against HBV infection.
The hepatitis B virus nucleocapsid plays a central role in the production of infectious xe2x80x9cDanexe2x80x9d particles. During the formation of the virus, the core particle must recruit into its structure the essential ingredients of viral replication, the pregenomic RNA and the viral reverse transcriptase. On its completion, the core particle must migrate to the endoplasmic reticulum, where the viral surface antigens and lipids are continuously assembled into envelope structures, and delivered to the extracellular environment via the secretory pathway. Thus, formation of the infectious virion requires the nucleocapsid to xe2x80x9ccapturexe2x80x9d the assembling surface proteins and pass through the secretory passageway.
Antibodies to proteins have been generated by immunization with short peptides having an amino acid sequence corresponding to the sequence of preselected protein fragments. Nima, et al, PNAS USA, 80: 4949-4953 (1983). Nevertheless, the generation of antibodies which recognize the native protein may depend on the appropriate conformation of the synthetic peptide immunogen, among other factors. Neurath et al., PNAS, 79:7871-7875 (1982). For this reason, immunization with synthetic peptide analogues of various virus proteins has only rarely resulted in production of virus-neutralizing antisera comparable to those elicited by the virus proteins themselves. Thus the preparation of synthetic immunogens mimicking antigenic determinants on intact viruses remains a challenge.
It has been suggested that HBV cores are not released from the cell without expression of envelope proteins, in contrast to the situation observed in retroviral assembly in general, where nucleocapsids can be exported in the absence of envelope gycoproteins. Bruss et al., PNAS, 88 1062-1063 (1991).
Certain vaccines have been described containing peptides with an amino acid chain of at least six consecutive amino acids within the pre-S gene coded region of the envelope of the hepatitis B virus. U.S. Pat. No. 5,204,096. However, these peptides do not appear to inhibit the assembly of the virus.
No safe and effective therapeutic treatment is presently available for hepatitis B infection, and clinical exploration of promising antiviral agents, such as nucleoside analogues, is hampered because of significant side-effects, resulting, for example, from their a specific body distribution.
Thus, there is a need for effective therapeutic and/or prophylactic agents against infection and diseases associated with HBV. The need has become even more urgent in view of the recent emergence of escape mutants of HBV that are not neutralized by vaccine induced antibodies.
Accordingly, the present invention is directed to novel peptides, and methods of treatment of diseases associated with HBV, which substantially obviate one or more of the problems due to the limitations and disadvantages of the related art. The peptides and small molecules taught and described herein are useful specifically for inhibiting the assembly of the HBV, thereby preventing disease and spread of infection.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the compositions, and methods particularly pointed out in the written description and claims hereof, as well as the appended drawings.
To achieve these and other advantages, and in accordance with the purpose of the invention, as embodied and broadly described herein, the invention relates to isolated, purified peptides which inhibit the assembly of the hepatitis B virus by binding to the core antigen of the virus, and thus prevent binding of the core antigen to the surface antigen. Specifically, the invention is directed to peptides which have a half maximal concentration (IC50) less than about 5, preferably less than 2, more preferably less than about 1, and most preferably less than about 0.5 xcexcM. Preferred peptides include, but are not limited to SLLGRMKG(xcex2-A)C (SEQ ID NO: 30), RSLLGRMKGA (SEQ ID NO: 31), HRSLLGRMKGA (SEQ ID NO: 32), MHRSLLGRMKGA (SEQ ID NO: 33), and RSLLGRMKGA(xcex2-A)C (SEQ ID NO: 34), or peptides derived therefrom.
In other embodiments, the invention is directed to compositions for inhibiting the assembly of the hepatitis B virus comprising the peptides described above. Further embodiments encompass methods of treatment and prevention, as well as pharmaceutical compositions such as vaccines.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.
Reference will now be made in detail to the presently preferred embodiments of the invention, examples of which are set forth in the accompanying drawings.
The present invention provides peptides and other small molecules which are useful in inhibiting the assembly of HBV virus. The invention also provides a means for identifying additional peptides or small molecules which, in combination or alone, are capable of inhibiting viral assembly. Furthermore, the invention provides peptides which may be useful in therapeutic and vaccinal compositions, as well as methods of making these compositions, and methods of treating infected individuals. In applicants previous work, peptide sequences that bind to the core antigen of hepatitis B virus (HBcAg) were identified by selection from a random phage display library and their affinities in solution determined in the phage associated form. The free peptide, ALLGRMKG (SEQ ID NO: 16), derived from the selected fusion phage sequences, was able to inhibit the interaction between the long hepatitis B virus surface antigen (L HBsAg) and HBcAg, with a half maximal concentration (IC50) of 10 xcexcM.
In an attempt to find improved inhibitors, a series of variants of the peptide ALLGRMKG (SEQ ID NO: 16) were provided by Drs. S. Adams and H. Cuervo (Biogen Inc.). However, none of these variants was an improvement over the initial sequence.
The present invention describes an alternative approach to the identification of improved inhibitors of L HBsAg binding to HBcAg, and encompasses peptides having a half maximal concentration (IC50) of less than about 5. Preferably, the peptides of the invention have an IC50 of less than about 2, more preferably, less than about 1, and most preferably, less than about 0.5 xcexcM.
As used herein, the term xe2x80x9cpeptidesxe2x80x9d refers to peptides, fragments, and analogs thereof, which, alone or in combination, are useful in inhibiting the assembly of the HBV virus. Preferably, the peptides are between about 2 to about 20 amino acids in length. More preferably, the peptides are between about 3 to about 12 amino acids in length. Such peptides can include the specific peptides identified in the tables below, as well as fragments and analogs thereof.
As used herein, the term xe2x80x9cfragmentxe2x80x9d refers to an amino acid sequence which is shorter than the peptide inhibitor from which it is derived, but which retains biological activity substantially identical to the original peptide. Preferably, such a fragment is at least two amino acids in length.
As used herein, the term xe2x80x9canalogxe2x80x9d refers to variations in the amino acid sequences of the inhibitor peptides, which may typically include analogs that differ by only one to about four amino acid changes. Other examples of analogs include peptides with minor amino acid variations from the inhibitors disclosed herein. In particular, peptides containing conservative amino acid replacements, i.e., those that take place within a family of amino acids that are related in their side chains, constitute analogs of the invention.
Genetically encoded amino acids are generally divided into four families: (1) acidic: aspartate, glutamate; (2) basic: lysine, arginine, histidine; (3) nonpolar: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar: glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In the present invention, for example, it is reasonable to expect that an isolated replacement of leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on its activity.
As used herein, the term xe2x80x9chomologous peptidexe2x80x9d includes peptide fragments which share at least 60 percent identity at the amino acid level, and preferably 75 percent identity, and substantially similar biological activity to a reference peptide. These preferred percentages reflect the small size of the peptides.
Peptide fragments which are particularly suited to the present invention are illustrated in Table 1.
The peptides, fragments, and analogs thereof, optionally associated with fusion partners, are preferably synthesized using conventional synthesis techniques, e.g., by chemical synthesis techniques. Alternatively, a skilled artesan may synthesize any of the peptides of the present invention by using an automated peptide synthesizer using standard chemistry such as, for example, t-BOC chemistry. (See L. A. Carpino, J. Am. Chem. Society, 79: 4427 (1957), the disclosure of which is hereby incorporated by reference.)
Alternatively, the peptides and other constructs of the invention may be prepared by known genetic engineering techniques, e.g., recombinant DNA techniques by cloning and expressing within a host microorganism or cell a DNA fragment carrying a coding sequence for the selected peptide or construct. Coding sequences for the peptides, fragments and fusion proteins can be prepared synthetically, or can be derived from viral RNA by known techniques, or from available cDNA-containing plasmids.
For use in the compositions and methods of the invention, it is anticipated that the above-described peptides, fragments and analogs thereof, may be designed into conventionally known, or alternative constructs to enhance production of the peptide or to enhance binding to HBcAg and thereby inhibit HBV assembly. For example, the peptides may optionally be fused to a protein or peptide fusion partner. Thus, one of skill in the art may design the peptide in association with a selected fusion partner, such as another peptide of the invention, or other peptides or proteins which impart desirable characteristics to the inhibitor peptide. Similarly, it is possible for one skilled in the art to engineer a peptide of the present invention as a fusion with an antibody or a fragment thereof.
Systems for cloning and expressing the peptides of the invention in various microorganisms and cells, including, for example, E-coli, bacillus, streptomyces, saccharomyces, mammalian, yeast and insect cells, and suitable vectors therefore, are known and available from private and public laboratories and depositories and from commercial vendors.
Whether produced recombinantly or synthesized, the peptides of the invention may be purified using conventional purification means. One of skill in the art can readily determine the appropriate level of purity required for the desired application for which the peptides are to be used.
These peptides and fragments of the invention are also useful as diagnostic reagents and vaccine components useful in the treatment of hepatitis B infection. The peptides and molecules disclosed herein may also be associated with a diagnostic label, a chemical marker, a toxin, or another protein or peptide, provided that the peptide associated with such a molecule is characterized by substantially the same biological activity as the original peptide.
The present invention also provides a means for identifying additional peptides which may inhibit the assembly of the hepatitis B virus. According to this method, one skilled in the art may also use HBsAg preparations to identify additional peptides which have analogous biological activity to the peptides disclosed herein, in that they also would inhibit the interaction between HBcAg and HBsAg, thereby preventing virus formation. Using techniques which are known to those skilled in the art, it is anticipated that the disclosure herein would enable one skilled in the art to identify other suitable peptides.
The peptides of the invention may be useful as diagnostic free agents, as well as therapeutics. Specifically, the peptides may be associated with conventional labels which are capable, alone or in combination with other compositions or compounds, of providing a detectable signal which would indicate the presence of the hepatitis B virus in a sample. A variety of enzyme systems have been described in the art which will operate to reveal, for example, a colorimetric signal in an assay, e.g., glucose oxidase, peroxide, tetramethylbenzadine systems (tmb), horse radish peroxidase (hrp) systems and other similar enzyme systems. Other label systems that may be utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles, magnetic particles, fluorescent compounds, radioactive compounds or elements, or immunoelectrodes.
Detectable labels for attachment to the peptides or constructs useful in the diagnostic assays of this invention may be easily selected from among numerous compositions known and readily available to one skilled in the art of diagnostic assays. The diagnostic methods and peptides of the invention are not limited by the particular detectable label or label system employed.
It will be understood by those skilled in the art that any number of conventional assay formats, particularly immunoassay formats, may be designed to utilize the peptides or constructs of the invention for the detection of HBV infection. This invention is thus not limited by the selection of the particular assay format, and is believed to encompass assay formats which are known to one of skill in the art. For convenience, reagents for assays according to this invention may be provided in the form of kits. These kits can include microtiter plates to which the peptides or constructs have been preabsorbed, various diluents and buffers, labeled conjugates for the detection of specifically bound peptides, and other signal generating reagents, such as enzyme substrates, cofactors and chromogens. Other components of these kits can easily be determined by one of skill in the art.
The present invention also provides compositions useful for therapeutic treatment of individuals infected with HBV or for vaccinations for preventing HBV infection. Such compositions comprise a peptide of the invention, a fragment or analog thereof, and may additionally contain pharmaceutically acceptable carriers or diluents suitable for administration for the treatment of such infections. Such compositions include a pharmaceutical preparation for the treatment of hepatitis B in mammals, said preparation comprising a therapeutically effective amount of a peptide which binds to HBcAg and interferes with the association of HBcAg and HbsAg; and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers will facilitate administration of the peptides, however, are physiologically inert and/or, nonharmful. Numerous carriers are known in the art and may be chosen based upon the desired application. Exemplary carriers include, but are not limited to, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, peptin, peanut oil, olive oil, sesame oil, and water. Additionally, the carrier or diluent may include a time delay material, such as glycerol monosterate or glycerol disterate, alone, or in combination with a wax. In addition, known slow release polymer formulations including, for example, soluble glasses, can be used.
In certain embodiments, a vaccine composition may contain a xe2x80x9ccocktailxe2x80x9d of multiple reagents useful in the treatment, or prevention, of hepatitis B infection. For example, a cocktail may include other reagents such as interferons, nucleoside analogs and/or N-acetyl-cysteine.
Optionally, the vaccine composition may further contain adjuvants such as conventional alum based adjuvants, or muramyl dipeptides, preservatives, chemical stabilizers or other antigenic proteins. Typically, stabilizers, adjuvants and preservatives etc. are optimized to determine the best formulation for efficacy in the desired application. Suitable preservatives may include chlorylbutynol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallade, parabens, glycerine, and phenol.
Suitable amounts of these compositions can be determined based upon the level of response desired. In general, the vaccine compositions may contain between 1 ng to 1000 mg of peptide. Suitable dosages of the vaccine composition of the invention can be readily determined as well. Generally, a suitable dose is between 0.1 to 5 milliliters of the vaccine composition. The dosage can also be determined readily by one skilled in the art based on the usual factors such as the weight, age, sex and/or general health of the patient being treated.
The present invention also provides a prophylactic method of administering to a subject an effective amount of the claimed composition. For example, for prevention of HBV infection, the claimed compositions may be administered as a vaccine which may be administered with a frequency which is dependent upon the likelihood of exposure to the virus. Where desirable, boosters may be co-administered. The vaccine may be administered by any suitable route, such as, for example parenteral administration, particularly intramuscular or subcutaneous, as well as oral administration. Thus, the present invention provides pharmaceutical compositions useful in providing passive immunity against infection by HBV.
The peptides claimed herein can be used in the active therapy of HBV infected individuals to inhibit, decrease, or slow the proliferation of the virus within the body. Therapeutic compositions comprise the claimed peptides capable of disabling, inhibiting or preventing the assembly mechanism of the virus. Such therapeutic compositions may be formulated to contain carriers or diluents, and one or more of the peptides of the invention. Such carriers and diluents are discussed above in connection with certain other compositions, and are readily identifiable by one of skill in the art. Optionally, the composition may contain other therapeutic agents useful against hepatitis B infection.
The peptides of the invention can be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid sequence coding for the peptide, or by chemical synthesis, or in certain limited situations, by chemical cleavage of a protein or other methods. When produced by recombinant techniques, host cells transformed with nucleic acids encoding the peptide are cultured in a medium suitable for the cells, and recombinant peptides are purified from the cell culture medium, host cells, or both, using techniques known in the art. The recombinant peptides of the invention are isolated such that the peptide is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when synthesized chemically, or obtained by chemical cleavage of a protein.
Preparation of vaccines which contain peptide sequences as active ingredients is well understood in the art. Typically, such vaccines are prepared as injectable vaccines, either as liquid solutions or suspension. Solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The preparation may in certain embodiments be emulsified or encapsulated in liposomes. The active ingredient can be mixed with any number of excipients which are pharmaceutically acceptable and compatible with the active ingredient or ingredients.